Chromosomal Insertion of Gfp Into Bacteria For Quality Control

ABSTRACT

An isolated mutated green fluorescent protein (gfp) gene for chromosomal insertion into a bacterium, wherein the gene is capable of being expressed in bacteria and produce sufficient fluorescence under illumination from a UV lamp in a bacterial colony to be seen by the naked eye. A gene cassette for inserting a gene into a chromosome.

TECHNICAL FIELD

The present invention is directed to methods for producing labelledcells, particularly visibly fluorescent bacteria suitable for use asquality control strains in microbiological or biological testing.

BACKGROUND

Microbiology testing laboratories maintain in-house culture collectionsof microorganisms for quality control purposes. These microorganisms areknown as quality control (QC) strains and are used as referencestandards and for quality control of the testing methods. Samples thatare known to be free of contamination are often spiked with a QC strainand this spiked sample is then processed through the test method. Apositive result from the spiked sample validates the testing method. TheQC strains are also used to quality control media that is used to growmicroorganisms. The media is inoculated with the QC strain and thegrowth is observed.

Traditionally these quality control checks that are performed with QCstrains are qualitative. Recently, however, regulatory authorities suchas ISO have begun enforcing quantitative quality control checks.

One problem that microbiology laboratories face is the issue of crosscontamination. Laboratories can inadvertently contaminate a real samplewith a QC strain. This results in a false positive result, which canhave enormous implications such as unnecessary product recalls orincorrect diagnosis of disease.

In order to help with identifying instances of cross contamination,laboratories try to use species of bacteria as QC strains that arerarely detected in their samples. For example, in Australia Salmonellasalford is used as a QC strain because it is rarely detected inclinical, food or environmental samples. When a laboratory detectsSalmonella, tests are performed to check that the Salmonella detected isnot Salmonella salford. If it does prove to be Salmonella salford thenthe validity of the result is questioned.

The use of rare species such as Salmonella salford as QC stains doeshelp to identify cross contamination problems, however, confirming theidentity of the strain that has been detected takes time. Typically,this confirmation takes between one and three days. In some instances,the confirmation has to be performed by a specialist laboratory. Theselengthy delays can have serious implications. For example, a productrecall may be delayed for several days during which time consumers wouldbe exposed to the risk of infection.

A further problem with the use of rare species as QC strains is that therare species may have biochemical or physiological properties that aredifferent to those of the commonly isolated organisms. For example,Salmonella salford does not grow well on some media that are routinelyused to isolate Salmonella from food, whereas the commonly isolatedSalmonella such as Salmonella typhyimurium do grow well on these media.Salmonella salford is therefore not a suitable QC strain for theseculture media.

In an attempt to address this problem, the present inventorshypothesized that it would be extremely useful to have QC microorganismsthat form colonies on agar plates are fluorescent when viewed by thenaked eye with illumination from a UV lamp.

The genetic modification of microorganisms with fluorescent genes hasbeen widely studied (GFP: Properties, Applications, and Protocols (1998)Chalfie M, Kain S. Wiley-Liss, New York, USA). The most commonlyemployed gene for a fluorescent protein is the green fluorescent protein(GFP) gene (gfp) from the jellyfish Aequorea Victoria. Genes encodingother fluorescent proteins have also been isolated from othercoelenterates.

Fluorescent bacteria have been created previously by incorporating a gfpgene into a plasmid and inserting the plasmid into the bacteria. Theplasmid normally contains an antibiotic resistant gene that allows thebacteria to be grown on antibiotic containing media. The antibioticskill any bacteria that do not retain the plasmid. Theseplasmid-containing strains only retain their fluorescence when grown onmedia that contain antibiotics.

An advantage of plasmid-carrying strains is that several hundred copiesof the plasmid are normally present within a bacterial single cell. Thismeans that several hundred copies of the fluorescence gene can be placedwithin each cell to create cells that are very fluorescent.

Plasmid instability can be a major problem in culturing bacteria,particularly if the cultures go through many generations by passaging.The resulting effects are loss of expression of any plasmid-encodedphenotype because of the build-up of non-productive plasmid-free cells.Plasmid instability can be due to segregational instability and/orstructural instability. Segregational instability is the loss of plasmidfrom one of the daughter cells during cell division because of defectivepartitioning. Structural instability is attributed to deletions,insertions and rearrangements in the plasmid DNA, resulting in the lossof expression of the encoded phenotype. Plasmid stability is influencedby the vector and host genotypes, vector copy number, and the origin andsize of foreign DNA have been observed to affect plasmid stability.Plasmid stability is also a function of physiological parameters thataffect the growth rate of the host cell, which include pH, temperature,aeration rate, medium components and heterologous protein accumulation.

Plasmid instability is undesirable in the production of bacterialstrains for quantitative QC methods, as consistent expression of the QCphenotype is paramount. Consistent expression could be achieved byirreversibly integrating the genes encoding the fluorescent phenotypeinto the host genome to ensure long-term stability and expression of thegene product. Ideally, only a single copy of the marker gene should beintegrated into the bacterial chromosome as this reduces the likelihoodof gene instability resulting from homologous recombination-mediatedgene excision.

The preferred requirement of a single copy fluorescence gene in thebacterial genome means that achieving sufficient fluorescence maybechallenging. In comparison, the use of a plasmid containing strainallows several hundred copies of the fluorescence gene to be present. Toensure that a high level of fluorescence is achieved with a single copyon the genome a transcriptional promoter should be chosen that ispowerful enough to produce visible fluorescence.

It has been found to be difficult to incorporate genes into a bacterialchromosome and still obtain the required selective or characteristicgenotype.

The present inventors have developed several strong bacterial promotersystems for the expression of fluorescent phenotypic markers inmicrobial cells.

SUMMARY OF INVENTION

The present inventors have devised methods which surprisingly allow forthe preparation of microorganisms that are fluorescent even whenpassaged multiple times on media that does not contain antibiotics orother selective pressures.

In a first aspect, the present invention provides an isolated mutatedgreen fluorescent protein (gfp) gene for insertion into the chromosomeof a bacterium, the gene is capable of being expressed and producesufficient fluorescence under illumination from a UV lamp in a bacterialcolony to be seen by the naked eye.

Mutations at nucleotides positions 1492, 1493 (Ser72Ala), 1737(Met153Thr) and 1766 (Val163Ala), as set out in FIG. 3, in the wild-typegfp gene, or mutations for synonymous codons which change the same aminoacid positions (Ser72Ala, Met153Thr and Val163Ala), are encompassed bythe present invention.

In a preferred form, the mutated gfp gene has a nucleic acid sequencefrom bases 1524 to 2240 as set out in FIG. 4 (SEQ ID NO 6).

The mutated gene can be preferably optimized for different bacteria. Thegene is particularly adapted for chromosomal insertion and expression inbacteria.

In a second aspect, the present invention provides an isolated greenfluorescent protein (GFP) expressed by the mutated gfp gene according tothe first aspect of the present invention.

In a third aspect, the present invention provides an isolated mutantgreen fluorescent protein (GFP) capable of producing sufficientfluorescence under illumination from a UV lamp in a bacterial colony tobe seen by the naked eye.

Preferably, the isolated mutant GFP is selected from a protein havingone or more mutations of mutant GFP01, mutant GFP02, mutant GFP03,mutant GFP07, mutant GFP10, mutant GFP15, mutant GFP16, mutant GFP20,mutant GFP21, mutant GFP22, mutant GFP26, mutant GFP27, mutant GFP37,mutant GFP43, mutant GFP44, mutant GFP53, mutant GFP54, or mutant GFP55as defined below in Table 2.

Preferably, the isolated mutant GFP is selected from mutant GFP01,mutant GFP02, mutant GFP03, mutant GFP07, mutant GFP10, mutant GFP15,mutant GFP16, mutant GFP20, mutant GFP21, mutant GFP22, mutant GFP26,mutant GFP27, mutant GFP37, mutant GFP43, mutant GFP44, mutant GFP53,mutant GFP54, or mutant GFP55 as defined below in Table 2.

In a preferred from, the isolated mutant GFP has an amino acid sequenceas set out in SEQ ID NO 4.

In a fourth aspect, the present invention provides an unrepressed orconstitutive gene cassette for providing a gene to a chromosomecomprising an endogenous gene under the control of the very strongbacteriophage lambda promoter left (P_(L)) and one or more transposonelements. It will be appreciated that the cassette can incorporate othersuitable transcriptional promoters to allow expression of a gene productin a cell or bacterium.

In one form, the cassette has a nucleotide sequence from 1 to 1278 and1996 to 2007 substantially as shown in FIG. 3, wherein the gene isinserted between positions 1728 and 1996.

In another form, the cassette has a nucleotide sequence from 1 to 1523and 2241 to 2326 substantially as shown in FIG. 4 (SEQ ID NO 4), whereinthe gene is inserted between positions 1523 and 2241.

In a preferred form, the cassette has a nucleotide sequence from 1 to309 and 1027 to 1032 substantially as shown in FIG. 5 (SEQ ID NO 7),wherein the gene is inserted between positions 310 and 1026 (SEQ ID NO8).

The cassette is suitable of inserting any gene, endogenous or exogenous,mutant or native into the chromosome of a bacterium or cell. Examplesinclude but not limited to genes encoding green fluorescent protein(gfp), red fluorescent protein, yellow fluorescent protein or anyunmodified or modified versions of known fluorescent proteins. Examplesalso include genes that encode proteins capable of catalysing theproduction of calorimetric or fluorescent pigments, including but notlimited to carotenoids, indole or indirubin.

Preferably, the gene is a green fluorescent protein (gfp) gene. Morepreferably, the gene is a mutant gfp gene.

The present inventors have demonstrated the suitability of the cassetteby developing bacteria having enhanced fluorescence by inserting a greenfluorescent protein (gfp) gene into the chromosome of the bacteria. Itwill be appreciated that the cassette can be used for any suitable geneto allow expression of the gene product in a cell or bacterium.

In a fifth aspect, the present invention provides a bacterium or cellcontaining a gene cassette according to the fourth aspect of the presentinvention.

In a sixth aspect, the present invention provides an unrepressed orconstitutive gene cassette for providing a green fluorescent protein(gfp) gene to a bacterium comprising a gfp gene under the control of astrong promoter and one or more transposon elements.

Preferably, the gene is under the control of the very strongbacteriophage lambda promoter left (P_(L)).

In one form, the cassette is substantially as shown in FIG. 3 (SEQ ID NO1).

In another form, the cassette is substantially as shown in FIG. 4 (SEQID NO 4).

Preferably, the cassette is as substantially as defined in FIG. 5 (SEQIN NO 7).

In a preferred form, the mutant gfp gene substantially as shown innucleotide bases 310 to 1026 of FIG. 5 (nucleotide bases 310 to 1026 ofSEQ ID NO 7).

The cassette is particularly suitable for providing a mutant greenfluorescent protein to the chromosome of a bacterium.

It will be appreciated that similar results could be achieved with otherforms of GFP, possibly even the wild type GFP would be sufficientlyfluorescent when used with the gene cassette according to the presentinvention.

In a seventh aspect, the present invention provides a bacterium or cellcontaining the mutated GFP gene according to the first aspect of thepresent invention or a gene cassette according to the fourth aspect ofthe present invention.

The bacterium can be any suitable bacterium such as Acinetobacterlwoffii, Aeromonas hydrophila, Aspergillus niger, Bacillus cereus,Bacillus subtilis, Campylobacter coli, Campylobacter jejuni, Candidaalbicans, Citrobacter freundii, Clostridium perfringens, Clostridiumsporogenes, Edwardsiella tarda, Enterobacter aerogenes, Enterobactercloacae, Enterococcus faecalis, Escherichia coli, Escherichia coli 0157,Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella aerogenes,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentus,Legionella pneumophila, Listeria innocua, Listeria ivanovii, Listeriamonocytogenes, Meth. Resist. Staph. Aureus, Neisseria gonorrhoeae,Proteus rettgeri, Proteus mirabilis, Proteus vulgaris, Pseudomonasaeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens, Rhodococcusequi, Salmonella abaetetuba, Saccharomyces cerevisiae, Salmonellasalford, Salmonella menston, Salmonella sofia, Salmonella Poona,Salmonella typhimurium, Salmonella poona, Serratia marcescens, Shigellasonnet, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcuspneumoniae, Streptococcus pyogenes, Vibrio parahaemolyticus, Yersiniaenterocolitica, Zygosaccharomyces rouxii.

Preferably, the bacterium is Escherichia col. More preferably, the E.coli is NCTC 9001 or NCTC 12241 as herein defined.

Preferably, the bacterium is Salmonella sp. More preferably, theSalmonella sp is Salmonella typhimurium or Salmonella abaetetuba asherein defined.

Preferably, the bacterium is Listeria sp. More preferably, the Listeriasp is L. monocytogenes.

Examples of bacteria containing a mutated GFP gene or cassette accordingto the present invention have been deposited with the AustralianNational Measurement Institute on 13 Sep. 2004 under Accession NosNM04/42817, NM04/42818, NM04/42819 and NM04/42820.

The identity of the deposited bacteria are as follows:

-   NM04/42817 is E. coli NCTC12241-   NM04/42818 is Salmonella abaetetuba-   NM04/42819 is Salmonella typhimurium-   NM04/42820 is E. coli NCTC9001

The cell can be any suitable cell including prokaryotic or eukaryotic.Examples of non-bacterial cells include fungal and yeast cells such asCandida albicans, Zygosaccharomyces rouxii or Aspergillus niger.

In a eighth aspect, the present invention provides a modified bacteriumcontaining a mutated gfp gene or cassette selected from NM04/42817,NM04/42818, NM04/42819 or NM04/42820.

In a ninth aspect, the present invention provides use of a bacteriumaccording to the seventh or eighth aspects of the present inventionhaving fluorescence as a detectable marker.

Preferably, the use is as a laboratory QC strain.

The fluorescent bacteria are suitable for use as internal qualitycontrols as described in WO 01/09281, incorporated herein by reference.

The fluorescent bacteria can be used for tracking purposes. Examples ofthis use include: studies on the transport of cells within theenvironment; tracking of cells within water treatment plants; studies ongene exchange in the environment.

The fluorescent bacteria can be used to leak test biological safetyequipment such as safety cabinets and respirators.

The fluorescent bacteria can be used to test the efficiency of anyprocess that is designed to remove or inactivate bacteria. Examplesinclude water filters, UV disinfection methods, chemical disinfectionmethods and heat treatment.

The fluorescent bacteria are also suitable for use in materials testingmethods. Examples of this include the testing of water fittings to showthat they do not support the growth of microorganisms.

The fluorescent bacteria are suitable for use in a sewage treatmentprocess. Sewage treatment relies on the presence of specific bacteriasuch as nitrifying bacteria. Adding fluorescent nitrifying bacteriawould allow accurate monitoring of cell density of nitrifying bacteriawithin the sewage treatment process.

Bacteria are commonly used to control non-desirable bacteria. Bacteriaare added to a process or a product to out-compete non-desirablebacteria. An example of this is the control of Salmonella on chickens.Chickens are routinely treated with Salmonella cells from a specificstrain of Salmonella, known as Salmonella sofia, that is not infectiousto humans. The Salmonella sofia colonises the chickens and out-competesmore harmful strains of Salmonella. At present there is not a simplemethod to check that the chickens are colonised with Salmonella sofia.By treating the chickens with a fluorescent form of Salmonella sofia thelevel of Salmonella sofia on a chicken or on processed chicken meatcould be easily monitored by measuring fluorescence. Fluorescencebacteria could be used for any process that requires the monitoring ofintroduced bacteria.

The bacterium can be supplied as a culture or in the form of a BioBall™(BTF Pty Ltd, Australia) according to U.S. Pat. No. 6,780,581 or WO03/020959.

The present inventors have found that the fluorescence is substantiallystable when the bacteria are passaged multiple times. Antibiotics, forexample, are not required to keep the fluorescence gene within thebacteria.

The mutated GFP gene results in brighter fluorescence in E. coli, forexample, compared with a wildtype GFP gene expressed in a plasmid in anequivalent strain of E coli. Not being bound by theory, the inventorsbelieve that brighter fluorescence may be due to rapid maturation of thegreen fluorescence protein in bacteria. As the mutations have slightlychanged the structure of the protein, it is likely that this changedstructure matures more rapidly in bacteria than the wild type GFP.

Although other mutants of the GFP gene have been made and disclosed inthe prior art, all these mutants have fluorescence excitation andemission properties different to the wild type GFP. The GFP mutantaccording to the present invention, however, has substantially the sameexcitation and emission spectra as the wild type GFP.

Although other forms of tagging bacteria on the chromosome have beenpublished previously, the tagging methods enable the bacteria to grow oncertain media or selective conditions.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia prior todevelopment of the present invention.

In order that the present invention may be more clearly understood,preferred embodiments will be described with reference to the followingdrawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A. Diagrammatic representation of oligonucleotide primer bindingpositions relative to the proposed gfp gene cassette. B. Diagrammaticrepresentation of the two PCR products generated with oligonucleotideprimer pairs as marked, for construction of the gfp gene cassette,containing the wild-type gfp gene, by overlap extension PCR. C.Diagrammatic representation of the five PCR products generated witholigonucleotide primer pairs as marked, for construction of the completegfp gene cassette, containing modified gfp genes, by overlap extensionPCR. D. Diagrammatic representation of the structure of the gfp genecassette showing the position and orientation of the clts857 and gfpgenes, and the positions of the lambda P_(R) and P_(L) operators. Therelative positions of the codons within gfp that are altered by sitedirected mutagenesis are marked by their corresponding amino acidchanges.

FIG. 2. A. Structure of the pEntransposon-CamR plasmid and the gfp genecassette. Transposon arms are labeled Mu. The pBR322 origin ofreplication is labeled ori. B. Structure of the plasmid generatedfollowing ligation to the gfp gene cassette into pEntransposon-CamR atEcoRI and SalI restriction sites. Arrows indicate relative bindingpositions and orientation of oligonucleotides PENTGWF and GFPGWR usedfor genomic walking PCR. C. Structure of the plasmid shown in B.following deletion of the clts857 gene and the lambda P_(R) promoter bydigestion with EcoRV and SmaI, followed by recircularisation byblunt-end ligation. Arrows indicate relative binding positions andorientation of oligonucleotides PENTGWF and GFPGWR used for genomicwalking PCR. D. Structure of the gfp gene cassette once excised from theplasmid shown in C. Arrows indicate relative binding positions andorientation of oligonucleotides PENTGWF and GFPGWR used for genomicwalking PCR.

FIG. 3 shows a sequence map of the gfp gene cassette (SEQ ID NO 1)containing the wild-type gfp gene, showing the location of the majorsequence elements as depicted in FIG. 1D. Key restriction sites areunderlined. The lambda P_(R) operators are underlined and marked R1-R3.The lambda P_(L) operators are underlined and marked L1-L3. Atranslation of the lambda cl^(ts857) gene is shown underneath the DNAsequence (running in reverse orientation 17 to 730 bp SEQ ID NO 2). Atranslation of the gfp gene is shown underneath the DNA sequence(running in forward orientation 1279 to 1995 bp; SEQ ID NO 3).

FIG. 4 shows a sequence map of the gfp gene cassette (SEQ ID NO 4)containing the modified gfp gene, after deletion of the lambdacl^(ts857) gene and lambda P_(R) promoter. Key restriction sites areitalicised. The lambda P_(L) operators are underlined and marked L1-L3.Transposon arms are underlined (6-54 bp and 2273-2321 bp). Codonschanged by site directed mutagenesis to generate the modified gfp geneare underlined and labelled. A translation of the chloramphenicolresistance gene is shown underneath the DNA sequence (278 to 937 bp; SEQID NO 5). A translation of the modified gfp gene is shown underneath theDNA sequence (1524 to 2240 bp; SEQ ID NO 6).

FIG. 5 shows a sequence map of the minimal gfp gene cassette (SEQ ID NO7) containing the modified gfp gene and lambda P_(L) promoter. Keyrestriction sites are italicised. The lambda P_(L) operators areunderlined and marked L1-L3. Codons changed by site directed mutagenesisto generate the modified gfp gene are underlined and labelled. Atranslation of the modified gfp gene is shown underneath the DNAsequence (310 to 1026 bp; SEQ ID NO 8).

MODE(S) FOR CARRYING OUT THE INVENTION EXAMPLE 1

The following example describes the creation of a GFP cassette, thecreation of mutants of GFP that showed increased fluorescence in aplasmid within E. coli and the chromosomal integration of the mutant GFPand the wild type GFP into E. coli.

Construction of a Temperature Controlled GFP Gene Cassette

In an attempt to obtain visibly detectable levels of GFP in bacterialcolonies, a gene cassette was constructed comprising a modified gfp geneunder the control of the very strong bacteriophage lambda promotersright and left (P_(R) and P_(L)), and the gene coding for lambdathermolabile cl^(ts857) repressor protein. The regulatory regions forthe gfp gene cassette were obtained from the controlled expressionvector pJLA602 (Schauder et al., 1987 Gene 52: 279-283) by PCR. Thecl^(ts857) repressor protein tightly represses transcription from thelambda P_(R) and P_(L) promoters when grown at temperatures below 37° C.Incubating growing cultures at 42° C. results in inactivation of thecl^(ts857) repressor protein resulting in high level transcription ofgenes placed immediately downstream of the lambda P_(L) promoter.

The modified gfp gene was constructed by site directed mutagenesis usingoverlapping sets of degenerate oligonucleotides to reconstruct the fulllength gfp gene with 6 modified codons positions. The modified sites andthe selected residue alterations were chosen for their proven ability toimprove the maturation and fluorescence of GFP in E. coli. The use ofdegenerate oligonucleotide primers resulted in the creation of a libraryof gfp genes that varied between 2 or 3 possible codons at each of the 6chosen codon positions. The oligonucleotide primers are described belowand are listed in Table 1. The binding positions of all oligonucleotideprimers with respect to the gfp cassette are shown in FIG. 1A. The PCRproducts generated with said primers for reconstruction of the gfpcassette is shown in FIG. 1C. The complete gfp cassette is shown in FIG.1D. The wild-type gfp gene was also placed into the cassette in the samemanner for use as a control (see FIG. 1B).

TABLE 1 Name Sequence SEQ ID NO gfpF0 5′-TTTTTTGAATTCTTATTTGTATAGTTCATC9 gfpR1 5′-CTTTACTCATGGCAGTCTCCAGTTTGT 10 gfpF15′-GAGACTGCCATGAGTAAAGGAGAAGA 11 gfpF25′-ATGGTSTTCAATGCTTTKCRAGATACCCAGATCA 12 TA gfpF35′-AACTATATYTTTCAAAGATGACGGGA 13 gfpF45′-CAAACAAAAGAATGGAATCAAAGYTAACTTCAAA 14 ATTAGA gfpR05′-TTTTTTGAATTCTTATTTGTATAGTTCATC 15 gfpR25′-AAAGCATTGAASACCATAMSMGAAAGTAGTGACA 16 AGT gfpR35′-CTTTGAAARATATAGTTCTTTCCTGTA 17 gfpR45′-TTCCATTCTTTTGTTTGTCTGCCRTGATGTATAC 18 ATTGTGT gfpEnt5′-CCAGTTTGCTCAGGCTCT 19 T7promppf15′-GGGGAATTCTTAATACGACTCACTATAGAAGGAG 20 ATATACATAT GGCCTCCGAGAACGTCATCARfppr1 5′-GGGGGGGTCGACCTACAGGAACAGGTGGTGG 21 PENTGWF5′-TGATCTTCCGTCACAGGT 22 GFPGWR 5′-GTAACAGCTGCTGGGATT 23

The oligonucleotides primers used for the PCR amplification of the gfpgene cassette regulatory regions were as follows:

-   gfpF0, 5′-TTTTTTGAATTCTTATTTGTATAGTTCATC-3′ (SEQ ID NO 9), a primer    to amplify bacteriophage lambda cl^(ts857)/P_(R)/P_(L) region from    pJLA602. This primer incorporates a SalI site for directional    ligation of the cassette into a plasmid vector.-   gfpR1, 5′-CTTTACTCATGGCAGTCTCCAGTTTGT-3′ (SEQ ID NO 10), a primer to    amplify and overlap the lambda cl^(ts857) repressor gene/P_(R)/P_(L)    with the gfp gene.

Variants of the GFP were generated by oligonucleotide directedmutagenesis of the wild type gfp gene. Mutagenic PCR amplifications ofthe GFP gene were performed using combinations of degenerate andnon-degenerate oligonucleotide primers.

For the site directed mutagenesis and PCR amplification of the gfp genethe following oligonucleotides were used:

-   gfpF1, 5′-GAGACTGCCATGAGTAAAGGAGAAGA-3′ (SEQ ID NO 11), a primer to    amplify and overlap lambda cl^(ts857)/P_(R)/P_(L)/atpE with the gfp    gene.-   gfpF2, 5′-ATGGTSTTCAATGCTTTKCRAGATACCCAGATCATA-3′ (SEQ ID NO 12), a    degenerate primer for the amplification and mutagenesis of gfp. This    primer introduces three possible point mutations, Ser65Ala or    Ser65Gly, Val68Leu, and Ser72Ala into the gfp gene.-   gfpF3, 5′-AACTATATYTTTCAAAGATGACGGGA-3′ (SEQ ID NO 13), a degenerate    primer for the amplification and mutagenesis of gfp. This primer    introduces a single possible point mutation, Phe99Ser, into the gfp    gene.-   gfpF4, 5′-CAAACAAAAGAATGGAATCAAAGYTAACTTCAAAATTAGA-3′ (SEQ ID NO    14), a degenerate primer for amplification and mutagenesis of gfp.    This primer introduces a single possible point mutation, Met153Thr,    into the gfp gene.-   gfpR0 primer 5′ TTTTTTGMTTCTTATTTGTATAGTTCATC-3′ (SEQ ID NO 15), a    primer to amplify the gfp gene. This primer incorporates an EcoRI    restriction site for directional ligation of the cassette into a    plasmid vector.-   gfpR2, 5′-AAAGCATTGAASACCATAMSMGAAAGTAGTGACAAGT-3′ (SEQ ID NO 16), a    degenerate primer for the amplification and mutagenesis of the gfp    gene. This primer introduces two possible point mutations, Ser65Ala    or Ser65Gly and Val68Leu, into the gfp gene.-   gfpR3 primer 5′-CTTTGAAARATATAGTTCTTTCCTGTA-3′ (SEQ ID NO 17), a    degenerate primer for amplification and mutagenesis of the gfp gene.    This primer introduces a single possible point mutation, Phe100Ser,    into the gfp gene.-   gfpR4 primer 5′-TTCCATTCTTTTGTTTGTCTGCCRTGATGTATACATTGTGT-3′ (SEQ ID    NO 18), a degenerate primer for amplification and mutagenesis of the    gfp gene. This primer introduces a single possible point mutation,    Met153Thr, into the gfp gene.

For amplification and mutagenesis of the gfp gene, plasmid DNA from thepGFP vector (Clontech) was used as template DNA. PCR amplification ofthe gfp gene, the regulatory regions and the gfp gene segments contained1× PCR Gene Amp buffer; 200 μM of each of the four deoxyribonucleotidetriphosphate (dATP, dTTP, dCTP, dGTP); 0.3 μM of each oligonucleotideprimer (forward and reverse); AmpliTaq-Gold Polymerase (AppliedBiosystems) and approximately 10 ng of template DNA in a 50 μl reactionvolume. PCR reactions were performed in a Gene Amp PCR system 2400(Applied Biosystems) programmed as follows: 95° C. for 10 min, (95° C.30 sec, 55° C. 30 sec, 72° C. 1 min) repeated for 30 cycles, and a finalcycle at 72° C. for 5 min. PCR products originating from amplificationreactions of the regulatory regions and the four different segments ofthe mutant gfp gene were visualized by agarose gel electrophoresis andthe DNA purified using a QIAquick gel extraction kit (Qiagen Inc).

Assembly of the gfp gene and regulatory regions into the gfp genecassette were performed by overlap extension PCR (Ho et al., 1989 Gene77:51-59). Optimization of PCR conditions was required for successfulassembly of the gfp gene cassette. Best results for the assembly of gfpgene segments into an full length gene were obtained by first performinga primerless overlap extension step with a programmed decrease in theannealing temperature (1° C. per PCR cycle) followed by addition ofoligonucleotides and a further PCR step. The PCR reagents used were thesame as that described above, except for combining and using the 4 gfpgene cassette segments, each at approximately 10 ng, as template DNA ina 50 μl PCR reaction. The relative positions of primers with respect tothe major elements in the GFP cassette are shown in FIG. 1A. Therelative positions of PCR products amplified and used for overlapextension reassembly of the GFP cassette are shown in FIG. 1B. The finalcomplete GFP cassette is depicted in FIG. 1C.

A PCR for the assembly of the wild type gfp gene and the upstream lambdaregulatory regions into a single cassette was performed as follows: aprimerless overlap extension stage (95° C. for 10 min, 95° C. 30 sec,50° C. down to 35° C. over 15 cycles with 1° C. decrease per cycle for30 sec each cycle), followed by addition of primers (gfpF0 and gfpR0)and a second PCR stage comprising 95° C.—30 sec, 55° C.—30 sec, 72° C.—1min, repeated for 30 cycles, and a final cycle at 72° C. for 5 min.

A PCR for the recombination of the four mutant GFP gene segments into apool of selectively mutated full length gfp genes was as follows: aprimeness overlap extension stage (95° C. for 10 min, 95° C. 30 sec, 50°C. down to 30° C. over 20 cycles with 1° C. decrease per cycle for 30sec each cycle), followed by addition of primers (gfpF1 and gfpR0) and asecond PCR stage of 95° C. 30 sec, 55° C. 30 sec, 72° C. 1 min, repeatedfor 30 cycles. After amplifying the assembled mutant gfp gene, a secondPCR reaction was carried out using the same conditions used for theassembly of the wild type gfp gene, but in this case using thereassembled mutant gfp gene and the upstream regulatory regions toreassemble the entire mutant gfp gene cassette.

The complete sequence of the mutated gfp gene cassette, including genetranslations, promoter elements, and mutation positions, is given inFIG. 3. Similarly, The complete sequence of the wild-type gfp genecassette is given in FIG. 4.

Creation of GFP Mutants

The gfp gene cassette was designed to include two unique restrictionenzyme sites, SalI at the 5′end and EcoRI at 3′end. These restrictionsites were used for direction ligation of the mutated gfp gene cassetteinto the multiple cloning site of the pEntranceposon CmR vector(Finnzymes). The pEntranceposon CmR vector is a high copy number plasmidconstructed by replacing the multiple cloning site of the high copynumber plasmid pUC19 with the bacterial phage Mu transposon, and thechloramphenicol resistance gene (CamR).

PCR product comprising the complete mutant gfp gene and the plasmidvector pEntranceposon CamR were digested with SalI and EcoRI restrictionenzymes (MBI Fermentas). The digested DNA fragments were visualized andpurified from agarose gel using a QIAquick gel Extraction kit (Qiagen).The digested mutated gfp gene cassette and pEntranceposon CamR vectorwere ligated together and transformed into E. coli DH5α to generate alibrary of clones containing gfp genes randomly mutated at 6 selectedpositions (see FIG. 2). Similarly, the wild type GFP gene cassette wasligated to the digested pEntranceposon CamR vector and transformed intoE. coli DH5 alpha.

Transformed cells were plated onto Luria Bertani (LB) agar media platescontaining 25 μg/ml of chloramphenicol and incubated at 28° C. After 48hours incubation, the incubation temperature was shifted to 42° C. fortwo to three hours for inactivation of the cl^(ts857) repressor proteinand induction of GFP expression. Colonies expressing GFP were screenedby visualization of fluorescence using a hand held ultraviolet lamp (UV365 nm). A number of colonies expressing GFP appeared on the plates.Colonies originating from the mutant GFP gene construct showed visuallydetectable variation in GFP fluorescence intensity emitted from coloniesand were graded accordingly.

Sequencing the Mutant GFP Clones

Five wild type GFP transformants and 18 mutant GFP transformants wereselected following visual screening for fluorescence. Several of themutant GFP transformants were significantly brighter than the wild typeGFP transformants. The selected mutant GFP clones included the brightestfluorescing colonies as well as colonies showing intermediate and lowintensity fluorescence. Single recombinant colonies were re-streaked tonew LB plates containing 25 μg/ml of chloramphenicol. These cells wereused for inoculation of 3 ml LB liquid media containing 25 μg/ml ofchloramphenicol. Cultures obtained after overnight incubation at 28° C.with shaking were used for plasmid DNA extraction using QIAprep kits(Qiagen). Extracted plasmid DNA from the selected GFP clones was thenused for nucleotide sequencing of the gfp gene cassette to determine thegfp genotype. Sequencing reactions (Big Dye terminator chemistry AppliedBiosystems Inc) were performed using plasmid DNA extracted from GFPtransformants. The oligonucleotide primers gfpF0 and gfpR1 were used forsequencing of the regulatory regions of GFP gene cassette, while gfpF1and gfpR0 primers for sequencing of the GFP gene open reading framewithin the GFP gene cassette. Sequencing results were analyzed using theGCG Wisconsin software package version 8 (Devereux J, Haeberli P,Smithies O, 1984, Nucleic Acids Res 12:387-395). Results from thenucleotide sequencing analysis were used for amino acid sequencealignment of wild type GFP and GFP mutants. A summary of amino acidchanges identified in the GFP mutants is presented in Table 2.

Results from the sequencing analysis indicated that the most frequentlyoccurring amino acid changes, Ser at position 72 to Ala, Met at position153 to Thr and Val at position 163 to Ala, were found in the GFP mutantsclones showing the brightest fluorescence intensity when visualized ascolonies on plates illuminated with ultraviolet light (UV 365 nm). Thesemutants were considerably brighter than colonies that contained the wildtype GFP.

The mutant gfp gene construct containing the three most frequent aminoacid changes as well as wild type GFP gene construct were chosen forbacterial chromosomal integration experiments as described below. Thisplasmid was named pENTcIGFP (See FIG. 2B).

TABLE 2 Modified amino acid residues Position 65 68 72 99 153 163 SerVal Ser Phe Met Val wild type GFP Ser Val Ser Phe Met Val mutant GFP01Ser Val Ala Phe Thr Ala mutant GFP02 Ala Val Ala Phe Thr Ala mutantGFP03 Ser Val Ser Phe Thr Ala mutant GFP07 Ser Val Ala Phe Thr Alamutant GFP10 Ser Val Ser Phe Thr Ala mutant GFP15 Ser Val Ala Phe ThrAla mutant GFP16 Ser Leu Ala Phe Thr Ala mutant GFP20 Ser Val Ala PheThr Ala mutant GFP21 Ser Leu Ala Phe Thr Ala mutant GFP22 Ser Val SerPhe Met Ala mutant GFP26 Ser Leu Ala Phe Thr Ala mutant GFP27 Ser LeuAla Ser Met Ala mutant GFP37 Ser Val Ala Phe Thr Ala mutant GFP43 SerVal Ser Phe Thr Ala mutant GFP44 Ser Leu Ala Ser Thr Ala mutant GFP53Ser Leu Ala Ser Met Ala mutant GFP54 Ser Val Ser Ser Met Val mutantGFP55 Ser Val Ser Ser Thr ValIntegration of GFP into the Chromosome of E. Coli

Integration of the gfp gene cassette into the E. coli DH5α genome wasperformed by insertion mutagenesis using bacteriophage Mu DNAtransposition complexes (Lamberg et al., 2002 Appl Environ Microbiol 68:705-712). This system was chosen as the transposon cassette does notinclude the gene encoding Mu transposase. Transposase enzyme is added topurified transposon DNA to form a transposition complex that is thentransferred into bacterial cells by electroporation. The transposasemediates the integration of the transposon into the genome, effectivelyresulting in irreversible integration of the transposon and any includedgenes, into the bacterial chromosome.

Plasmid DNA of pEntranceposon vectors containing the GFP gene cassettewere used for BglII restriction enzyme digests. The BglII digestionexcises the transposon from the pEntranceposon vector. Digested plasmidDNA was separated using agarose gel electrophoresis and thepEntranceposon CamR transposon fragment containing the gfp gene cassettepurified using a QIAquick gel Extraction kit (Qiagen Inc). PurifiedpEntranceposon DNA was used for transpososome assembly reactions.

Transpososomes are stable protein DNA complexes formed by the binding ofMuA transposase protein into specific binding sites at each end of thetransposon DNA.

Transpososome formation reactions were optimized by titration of theamount of pEntranceposon DNA against a fixed amount of MuA transposaseenzyme (Finnzymes).

Reagents for transpososome assembly reaction mixtures (20 μl) included˜6 pmol of MuA transposase, 50% glycerol, 150 mM of Tris-HCl (pH 6.0),150 mM NaCl, 0.1 mM EDTA, and 0.025% (v/v) Triton X-100. Transpososomereactions were performed by adding ˜0.25 pmol to ˜1.0 pmol ofpEntranceposon DNA containing the GFP gene cassette to the mixturesfollowed by incubating at 30° C. for 2-3 hours. Transpososome formationwas visualized by electrophoresis on 2% agarose-TAE buffer gelcontaining 80 μg/ml of bovine serum albumen. Transpososome assemblyreaction samples (1 μl) were loaded into the gel using 0.2 (v/v) ofFicoll 400 as loading buffer. After gel visualization, selectedtranspososome complexes were used for the transformation ofelectrocompetent E. coli DH5α cells.

Electrocompetent E. coli cells were prepared by growing 500 ml ofculture in SOB medium at 37° C. with shaking to optical density at 600nm of 0.8. Cells were then harvested by centrifugation at 4° C. andresuspended in 25 ml of ice-cold 10% glycerol four times consecutively,then resuspended in 1 ml of ice-cold 10% glycerol. Aliquots of 1 μl oftranspososome reactions were used for electroporation of 40 μlelectro-competent E. coli cells using a Genepulser (Bio-Rad) at thefollowing settings: voltage 2.5 kV; capacitance 25 μF; resistance 200Ω(Bio-Rad 2-mm electrode spacing cuvettes). After electroporation 1 ml ofSOC medium was added, incubated for 90 minutes, spread onto LB platescontaining 25 μg/ml of chloramphenicol and incubated at 28° C. After 48hours incubation at 28° C. cultures incubation temperature was shiftedto 42° C. for two to three hours to induce GFP expression.

The few colonies that appeared on plates originated from integration ofboth mutant and wild type GFP gene cassette showed no fluorescence whenilluminated with UV lamp. To verify if the integration of the GFP genecassette was successful, PCR amplification reactions using gfpF1 andgfpR0 primers were carried using colonies growing on plates originatingfrom electro-transformation of both mutant and wild type GFPtranspososomes. Results were positive for the presence of the GFP genein the colonies. Four selected positive colonies (both mutant and wildtype GFP putative integrants) were grown overnight in liquid media(LB+chloramphenicol). After harvesting cultures by centrifugation,genomic DNA from ˜100 μg of cell paste was extracted using DNAextraction kit (Fast DNA, BIO 101 systems). Genomic DNA from twodifferent isolates originating from both mutant and wild type GFPputative integrants were used for nucleotide sequencing using the systemof analysis as described above. Oligonucleotide primers gfpF0 gfpR1 andgfpF1 gfpR0 were used for sequencing of both regulatory and open readingframe within the GFP gene cassette.

The above results confirmed the integration of intact GFP gene cassettesequence in the genomic DNA of the putative integrants. However, theywere not visibly fluorescent when grown under inducing conditions. Itwas considered that this negative result might be due to the single copynature of the integrated gfp gene in combination with the non-idealgrowth temperature when inducing protein production at 42° C. Usually,the lambda cl^(ts857)/P_(R)/P_(L) system is used to induce proteinproduction in high copy number plasmid systems, with maximum proteinyields usually obtained within 2-3 hours of switching to 42° C. However,with GFP being produced from only a single copy gene we considered thata time potentially much longer than 2-3 hours might be required toachieve visible levels of GFP, and that the 42° C. growth conditionsmight prevent continued expression of GFP to visible levels. Therefore,we considered that removal of the cl^(ts857) gene and the P_(R) promotermight allow unrepressed constitutive high-level expression of the GFPprotein from the remaining P_(L) promoter. A simple deletion strategywas devised to test this hypothesis (see Example 2).

EXAMPLE 2

The temperature inducible gfp gene cassette integrated in E. coliresulted in non-fluorescing colonies. As discussed above, it wasconsidered that a prolonged growth at the 42° C. induction temperaturemay have inhibited protein production in E. coli before detectableamounts of GFP could be produced from a single copy gfp gene. Thefollowing procedure was devised for the deletion of the cl^(ts857) genefrom the plasmid to create an unrepressed (constitutively expressed)version of gfp gene cassette.

Generation of Unrepressed gfp Gene Cassette

Analysis of the nucleotide sequence of gfp gene cassette revealed aunique SmaI restriction enzyme that if used in combination with an EcoRVsite in the pEntcIGFP plasmid (see FIG. 2C) would result in excision ofmost of the cl^(ts857) gene and P_(R) from the cassette. Excision of thecl^(ts857) gene would effectively promote high-level constitutivetranscription of gfp from the P_(L) promoter (see FIGS. 2C and 2D).

The pEntcIGFP plasmid containing the gfp gene cassette was restrictiondigested with SmaI and EcoRV (MBI Fermentas). The digested DNA wasvisualized by agarose gel electrophoresis and DNA corresponding to thevector minus excised cl^(ts857) gene was gel purified using a QIAquickgel extraction kit (Qiagen Inc). The digested DNA was ligated blunt-endusing T4 DNA ligase (Roche) at 14° C. for 12 hr. The ligated DNA wasused to transform chemically competent E. coli DH5 alpha cells, platedonto LB plates containing chloramphenicol and incubated overnight at 37°C. Positive colonies expressing GFP (following visual inspection underUV light) appeared on plates originating from a ligation of the mutantGFP gene cassette with the deleted cl^(ts857) repressor gene. Thepositive transformants were re-streaked to individual plates and singlescolonies used for liquid cultures in LB medium containingchloramphenicol. After incubation overnight at 37° C., an aliquot fromthe culture was then used for plasmid extraction using QIAprep plasmidpurification kit (Qiagen). Excision of the cl^(ts857) gene from the GFPgene cassette was confirmed after analysing an EcoRI restriction enzymedigest of the extracted plasmid by agarose gel electrophoresis. Theplasmid containing the GFP gene cassette with the deleted cl^(ts857)gene was named pEntPLGFP, and the modified cassette is from hereinreferred as unrepressed GFP gene cassette.

Chromosomal Integration of Unrepressed GFP Gene Cassette into E. Coliand Salmonella Strains

Bacteriophage Mu DNA transposition complexes derived from pEntPLGFP wasused for chromosomal integration of the unrepressed gfp gene cassetteinto E. coli and Salmonella strains. E. coli DH5 alpha and E. coli NCTC12241 and E. coli NCTC 9001, and Salmonella typhyimurium and Salmonellaabaetetuba cells were transformed as follows.

pEntPLGFP plasmid DNA was restriction digested with BglII (see FIG. 2).The resulting two restriction fragments were separated by gelelectrophoresis and the DNA fragment comprising the Mu transposon andunrepressed mutant gfp gene cassette purified using a Qiaquick gelextraction kit (Qiagen).

Transpososome assembly reaction mixtures (20 μl) consisted of ˜6 pmol ofMuA transposase (Finnzymes), 50% glycerol, 150 mM of Tris-HCl (pH 6.0),150 mM NaCl, 0.1 mM EDTA, and 0.025% (v/v) Triton X-100, and ˜0.1 pmolto ˜0.9 pmol of pEntranceposon DNA containing the unrepressed gfp genecassette.

Transpososome formation reactions were incubated at 30° C. for 2-3hours, visualized on 2% agarose-TAE electrophoresis as described aboveand used for the transformation of E. coli NCTC 12241, E. coli NCTC9001, Salmonella typhyimurium and Salmonella abaetetuba electrocompetentcells. Electrocompetent E. coli and Salmonella cells were all preparedand electroporated using the method described above. Afterelectroporation 1 ml of SOC medium was added, incubated for 90 minutes,spread onto LB plates containing 25 μg/ml of chloramphenicol andincubated at 37° C. After 12 to 16 hours of growth at 37° C., coloniesexpressing the GFP protein could be easily visualized by illumination ofplates with a hand held UV light.

As the gfpF0 primer binding site was deleted from the gfp gene cassetteduring deletion of the cl^(ts857) gene, a new oligonucleotide primer,gfpEnt 5′-CCAGTTTGCTCAGGCTCT-3′ (SEQ ID NO 19), was synthesized for PCRamplification the unrepressed gfp gene cassette. PCR amplification ofthe gfp cassette using gfpEnt and gfpR0 primers were performed usingchloramphenicol resistant colonies obtained from electro-transformationwith the unrepressed gfp cassette transpososome. Correct sized PCRproducts indicated the presence of the unrepressed gfp gene cassette inall tested colonies. Four positive colonies from each of each E. coliand Salmonella strains were selected and grown overnight in liquid media(LB+chloramphenicol). Genomic DNA was then extracted from the cellsusing a DNA extraction kit (Fast DNA, BIO 101 systems). PCR productamplified from genomic DNA from two different transformants of each E.coli and Salmonella strain was used for nucleotide sequencing. Allsequencing results confirmed the presence of the unrepressed gfp genecassette in the genomic DNA of both E. coli and Salmonellatransformants. These transformants were clearly fluorescent when grownon a range of agar plates and illuminated with a UV light.

EXAMPLE 3

Identification of the Insertion Points of the Unrepressed gfp GeneCassette into E. Coli and Salmonella Integrant Chromosomes

Genome walking PCR (GWPCR) was used for identification of the insertionpoints of the unrepressed gfp gene cassette into E. coli and Salmonellastrains. Two genome walking primers were designed to walk upstream anddownstream of the gfp gene cassette. Primer PENTGWF was used to genomewalk from the 5′ end of the gfp gene cassette and GFPGWR to walk fromthe 3′ end of the cassette insertion point (See Table 1, FIGS. 2C and2D).

Genomic walking PCRs and synthetic DNA linker assemblage were carriedout according to the method described by Morris et al, Appl EnvironMicrobiol (1995) 61:2262-2269. The PENTGWF and GFPGWR primers were usedin combination with primers complementary to the generic linker forGWPCR reactions. PCR products ranging from 300 bp to 800 bp weresequenced and results used for match searches in bacterial genomesdatabase for identification of gfp gene cassette insertion points andflaking regions.

Insertion points of gfp gene cassette on the genomes of E. coli andSalmonella integrants were identified based on the published genomesequence data on Salmonella typhimurium and E. coli K12. For thefluorescent E. coli EC11775 strain, the gfp gene cassette was observedto be inserted into a gene encoding a homolog of E. coli K12 Zincbinding periplasmic protein (ZnaP). For the fluorescent E. coli BL21,the gfp gene cassette was inserted into a gene encoding 16S rRNA. Forthe fluorescent Salmonella abaetetuba, the gfp gene cassette wasinserted into a gene homolog of S. typhimurium ATP-dependent helicaseprotein (hrpA). In the fluorescent Salmonella typhimurium, the gfp genecassette was inserted into the sequence of a gene encoding a commonantigen found in the outer membrane of Salmonella and otherenterobacteria.

EXAMPLE 4 Generation of Fluorescent Listeria Monocytogenes

The strategy used to generate fluorescent Listeria monocytogens followedsimilar approach to that used for the E. coli and Salmonella strains.First, the gfp gene mut1 in an E. coli/Listeria shuttle vector pNF8vector (Fortineau et al., 2000 Res Microbiol 151: 353-360) was replacedwith our triple mutant gfp gene (gfp mut1 is a FACS optimized GFP withshifted excitation wavelength up to 488 nm). The gfp gene present inpEntPLGFP was amplified by PCR using the primers detailed in Table 3.

TABLE 3 SEQ ID Name Sequence NO Gfpmutf15′-AAACGGGATCCGAAAGGAGGTTTATTAAAATGAG 24 TAAAGGAGAAGAACTT Gfpmutr15′-AAAAAACTGCAGTTATTTGTATAGTTCATCCATG 25 CCAThe gfpmut1F primer was designed to introduce a BamHI restriction enzymesite and a consensus gram-positive ribosome binding site and the reversegfpmt1R primer was designed to introduce a PstI site in the PCRproducts. The resulting PCR product and the pNF8 were digested withBamHI and PstI restriction enzymes (MBI Fermentas), ligated using T4ligase (Roche) and used to transform chemically competent E. coli DH5αcells. Recombinant cultures harbouring the newly generated plasmidvector were recovered from selective LB agar plates containing 150 μ/mLof erythromycin (selective antibiotic resistance encoded in pNF8vector). After incubation at 30° C. over 72 hr visibly green coloniesappeared on plates (GFP expression in those recombinant clones in drivenby the Listeria Pdlt promoter located just upstream of the gfp gene inthe vector). Three putative recombinant clones were streaked onto freshLB+erythromycin plates and single colonies used to inoculateLB+erythromycin liquid media followed by overnight incubation at 37° C.with shaking. In order to verify if the isolated pNF8 plasmid wascarrying our triple mutant gfp gene and not the original FACS optimizedGFP gfp gene mut1, an aliquot of the cultures were used for fluorescenceassay measurements using a FLUOstar fluorimeter (BMG Lab TechnologiesGmbH). Two different excitation wavelengths—360 nm and 480 nnm withfixed 520 nm emission were tested. Results showed high fluorescence fromcells bearing the original pNF8 plasmid at 480 nm and lower fluorescenceat 360 nm excitation, whereas opposite results for cells bearing therecombined triple mutant gfp with high fluorescence at 360 nm and lowerfluorescence at 480 nm. One selected recombinant bearing thereconstructed vector named pNFMT1 was used for inoculation of LB liquidmedia containing 150 μl/mL erythromycin and incubated overnight at 37°C. The culture obtained was used for plasmid extraction using QIAprepkit (Qiagen).

L. monocytogenes electrocompetent cells were prepared by growing 250 mlof culture in brain and heart infusion (BHI) media containing 0.5 M ofsucrose with shaking at 37° C. to optical density measured at 600 nm of0.2. Penicillin was then added to 10 μl/ml and the culture grown tooptical density measured at 600 nm of 0.5. Cells were cooled on ice andharvested by centrifugation at 4° C. and resuspended in 100 ml of icecold 0.5 M sucrose in 1 mM HEPES pH 7.0 three times consecutively, thenresupended in 1 ml of ice cold 0.5 M sucrose in 1 mM HEPES pH 7.0, andstored as 50 μl aliquots at −80° C. Transformation efficiency of L.monocytogenes electrocompetent cells was evaluated using the pNFMT1vector. Enumeration of fluorescent L. monocytogenes colonies LB agarplates containing 150 μL/mL of erythromycin indicated transformationefficiencies up to 10̂5 c.f.u. per μg of pNFMT1 DNA.

The Pdlt promoter-mutant gfp gene construct present in the pNFMT1 wasexcised from the vector by restriction digest using EcoRI and Hind IIIrestriction enzymes (MBI Fermentas). The pEntranceposon vector(Finnzymes) was also digested with EcoRI and Hind III restrictionenzymes (MBI Fermentas). DNA corresponding to the digested Pdltpromoter-mutant gfp gene and the digested pEntranceposon vector wereseparated by agarose gel electrophoresis and purified from gels using aQIAquick gel extraction kit (Qiagen). The digested DNA was used forligation using T4 ligase (Roche) for generation of the plasmid vectorpEnt-Pdlt-GFPMT1. The pEnt-Pdlt-GFPMT1 vector was then used to transformcompetent E. coli DH5α cells. Recombinant cultures harbouring the vectorpEnt-Pdlt-GFPMT1 were recovered from selective LB agar plates containing25 μl/ml of chloramphenicol (selective antibiotic resistance encoded inpEnt-Pdlt-GFPMT1 vector).

The pEnt-Pdlt-GFPMT1 vector was digested with BglII restriction enzymeand the excised CamR transposon fragment containing the Pdlt-gfpseparated by agarose gel electrophoresis and purified using a QIAquickgel Extraction kit (Qiagen Inc). Purified CamR Pdlt-gfp transposon DNAwas used for transpososome assembly reactions. Transpososome formationreactions were optimized by titration of the amount of CamR Pdlt-gfpgene transposon DNA against a fixed amount of MuA transposase enzyme(Finnzymes). Reagents for transpososome assembly reaction mixtures (20μl) included ˜6 pmol of MuA transposase, 50% glycerol, 150 mM ofTris-HCl (pH 6.0), 150 mM NaCl, 0.1 mM EDTA, and 0.025% (v/v) TritonX-100. Transpososome reactions were performed by adding ˜0.25 pmol to˜1.0 pmol of Pdlt-gfp gene transposon DNA to the mixtures followed byincubating at 30° C. for 2-3 hours. Transpososome formation wasvisualized by electrophoresis on 2% agarose-TAE buffer gel containing 80μg/ml of bovine serum albumen. Transpososome assembly reaction samples(1 μl) were loaded into the gel using 0.2 (v/v) of Ficoll 400 as loadingbuffer. After gel visualization, selected transpososome complexes wereused for the transformation of L. monocytogenes electrocompetent cells.Aliquots of 1 μl of transpososome reactions were used forelectroporation of 40 μl electrocompetent L. monocytogenes cells using aGenepulser (Bio-Rad) at the following settings: voltage 2.5 kV;capacitance 25 μF; resistance 200Ω (Bio-Rad 2-mm electrode spacingcuvettes). After electroporation 1 ml of BHI medium was added, incubatedfor 90 minutes, spread onto LB plates containing 25 μg/ml ofchloramphenicol and incubated at 30° C.

EXAMPLE 5

This section describes the expression of another fluorescent protein, ared fluorescent protein known as DsRed2, in E. coli. The Dsred2 wassuccessfully integrated onto the chromosome of E. coli but nofluorescence was visible when examined under a UV light.

E. Coli Containing Red Fluorescent Protein on a Plasmid

A gene cassette was constructed by placing T7 promoter and ribosomalbinding site upstream of starting codon of DsRed2 gene (Clontech) usingthe primers T7promppf1 and Rfppr1 (see Table 1). The cassette, hereinreferred to as the T7DsRed2 gene cassette, was constructed then ligatedinto the pEntranceposon plasmid (Finnzymes). This plasmid wassubsequently transformed into E. coli DH5 alpha cells.

Colonies of the plasmid containing E. coli showed a bright redfluorescence after two days of growth on agar containingchloramphenicol. Only weak fluorescence was observed after 24 hoursgrowth.

Chromosomal Integration of Red Fluorescent Protein in E. Coli

The pEntranceposon-CmR plasmid containing the T7-DsRed2 gene cassettewas digested by BglII restriction enzyme digest and the releasedtransposon used for MuA transpososome formation (Finnzymes). Aliquots ofthe transpososome formation were used for transformation ofelectrocompetent E. coli BL21 (DE3) cells and chloramphenicol resistanttransformants were recovered after incubation on plates for 18-24 hoursat 37° C. Four transformants were isolated, chromosomal DNA extractedand analyzed for incorporation of T7DsRed2 gene into the chromosome byPCR. Results were positive for the clones tested, indicating theincorporation of T7DsRed2 gene into the genome of cultures. For one ofthe clones nucleotide sequencing was performed and the T7DsRed2 genesequence was confirmed to be intact. Transformants were cultured underinducing conditions on media containing 1 mM IPTG, an inducer oftranscription from the T7 promoter. However, no DSRed2 fluorescencecould be detected from cultures when plates were illuminated with UVlight.

EXAMPLE 6 Stability of Fluorescent Strains

The fluorescence of the E. coli, Listeria and Salmonella integrantstrains were examined to ensure that these organisms remainedfluorescent when passaged multiple times.

Yeast extract broth was inoculated with each of the fluorescent E. coliand Salmonella strains. A broth of Brain Heart Infusion (Oxoid) wasinoculated with the fluorescent Listeria strain. The cultures wereincubated with shaking at 37° C. for 24 hours. A loopfull of culture wasthen streaked onto nutrient agar or in the case of Listeria onto bloodagar and incubated at 37° C. for 24 hours. The plates were thenilluminated with a UV light and carefully examined for the presence ofnon-fluorescent colonies.

A colony from each plate was then used to inoculate another broth andthe entire process was repeated. This continued for 10 rounds ofculturing.

No non-fluorescent colonies were observed. An atypical fluorescentcolony was observed with the Salmonella abatetuba culture. The colonyappeared less smooth and flatter than the normal colonies for thisstrain. Biochemical analysis revealed that it was Salmonella abatetubabut a mutant that formed atypical colonies. It is likely that therepeated exposure to UV light caused the mutation. The mutant was stillfluorescent.

These results demonstrate that the fluorescence gene is highly stable inthese strains of bacteria.

SUMMARY

The present inventors have determined that three select modifications tothe GFP protein, namely S72A, M153T and V163A, result is consistentlyhigher visible expression of the protein when expressed in E. coli in aplasmid.

It has been determined that using the unrepressed lambda P_(L) promoter,it is possible to constitutively express visibly detectable levels ofthe modified form of GFP from a single copy of the gfp gene integratedinto the genome of 3 different strains of E. coli and two species ofSalmonella. Initial experiments indicated that expression was stablethrough long periods of growth and cell division. Precise genomicintegration points were determined for each of the E. coli andSalmonella gfp-cassette integrants.

Detectable levels of the modified GFP were observed when expressed inListeria monocytogenes under the control of the Listeria Pdlt promoter.

Detectable levels of the modified GFP were not observed when expressedfrom a single copy integrated gene under the control of the lambdaci^(ts857) repressor protein. Similarly, the fluorescent protein DsRed2could not be detected when expressed from a single copy integrated geneunder the control of a T7 RNA polymerase/T7 promoter system.

Means for the production of cells visibly altered by expression of amodified GFP protein have been developed that make them useful as QCstrains in microbiological testing.

It should be appreciated that this technique is applicable to otherbacterial species, and that other promoter systems and marker genescould be used in a similar fashion to achieve visible alteration ofbacterial cells.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1-24. (canceled)
 25. An isolated mutated green fluorescent protein (gfp)gene having mutations at nucleotides positions 1492, 1493 (Ser72Ala),1737 (Met153Thr) and 1766 (Val163Ala) in the wild-type gfp gene as setout in SEQ ID NO 1, or mutations for synonymous codons which change thesame amino acid positions (Ser72Ala, Met153Thr and Val163Ala).
 26. Theisolated gene of claim 25 having a nucleic acid sequence from bases 1524to 2240 as set out in SEQ ID NO
 4. 27. An isolated green fluorescentprotein (GFP) expressed by the mutated gfp gene according to claim 25.28. The isolated mutant GFP according to claim 25 comprising one or moremutations of mutant GFP01, mutant GFP02, mutant GFP03, mutant GFP07,mutant GFP10, mutant GFP15, mutant GFP16, mutant GFP20, mutant GFP21,mutant GFP22, mutant GFP26, mutant GFP27, mutant GFP37, mutant GFP43,mutant GFP44, mutant GFP53, mutant GFP54, or mutant GFP55.
 29. Theisolated mutant GFP according to claim 27 comprising mutant GFP01,mutant GFP02, mutant GFP03, mutant GFP07, mutant GFP10, mutant GFP15,mutant GFP16, mutant GFP20, mutant GFP21, mutant GFP22, mutant GFP26,mutant GFP27, mutant GFP37, mutant GFP43, mutant GFP44, mutant GFP53,mutant GFP54, or mutant GFP55.
 30. The isolated mutant GFP according toclaim 29 having an amino acid sequence as set out in SEQ ID NO
 6. 31. Abacterium or cell containing the mutated gfp gene according to claim 25.32. The bacterium according to claim 31 selected from the groupconsisting of Acinetobacter lwoffii, Aeromonas hydrophila, Aspergillusniger, Bacillus cereus, Bacillus subtilis, Campylobacter coli,Campylobacter jejuni, Candida albicans, Citrobacter freundii,Clostridium perfringens, Clostridium sporogenes, Edwardsiella tarda,Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis,Escherichia coli, Escherichia coli 0157, Haemophilus influenzae,Klebsiella pneumoniae, Klebsiella aerogenes, Lactobacillus acidophilus,Lactobacillus casei, Lactobacillus fermentus, Legionella pneumophila,Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Meth.Resist. Staph. Aureus, Neisseria gonorrhoeae, Proteus rettgeri, Proteusmirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonascepacia, Pseudomonas fluorescens, Rhodococcus equi, Salmonellaabaetetuba, Saccharomyces cerevisiae, Salmonella salford, Salmonellamenston, Salmonella sofia, Salmonella Poona, Salmonella typhimurium,Salmonella poona, Serratia marcescens, Shigella sonnei, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae,Streptococcus pyogenes, Vibrio parahaemolyticus, Yersiniaenterocolitica, and Zygosaccharomyces rouxii.
 33. The bacteriumaccording to claim 32 being Escherichia coli, Salmonella sp or Listeriasp.
 34. The bacterium according to claim 33 wherein the Salmonella sp isSalmonella typhimurium or Salmonella abaetetuba and the Listeria sp isL. monocytogenes.
 35. The cell according to claim 31 comprising Candidaalbicans, Zygosaccharomyces rouxii or Aspergillus niger.